Particular microorganisms have long been recognized as a source of skin infectious diseases. Pathogenic microorganisms cause infections by disrupting the normal functions of a host. Many pathogenic microorganisms, including intracellular bacteria, parasites, pathogenic yeast, and enveloped viruses, grow primarily in host cells where they are shielded from the effects of both antibodies and cytotoxic T cells. By developing ways to avoid the immune system, such microorganisms are able to multiply, and subsequently cause or contribute to inflammation and tissue damage in the infected organism.
As an example, tuberculosis (TB), caused by exposure to and infection with the mycobacterium, Mycobacterium tuberculosis, continues to infect and kill approximately 2 million people each year worldwide. It is estimated that one out of three humans are infected, leading to 8,000,000 new cases of active tuberculosis each year (Gye et al., Jama, 2822677-86, 1999). Greater knowledge of the mechanisms of human resistance to this pathogen as well as new therapeutics are needed. One of the first cell types to encounter M. tuberculosis after inhalation of the organism is the macrophage.
However, M. tuberculosis multiplies rapidly in cultured human macrophages even when they are stimulated with cytokines (Douvas et al., Infect Immun., 5021-8, 1985). Therefore, other elements of the immune system may assist macrophages in limiting the multiplication of tuberclebacilli in approximately one third of the earth's human population which is infected with M. tuberculosis, but does not develop active disease (Dye et al., Jama, 2822677-86, 1999).
Antimicrobial peptides are a recently discovered component of the innate immune system. They have been described in plants, tunicates, insects, fish, amphibia, and mammals, including humans, and are proposed to participate in the early host defense response against microorganisms.
They are likely to be particularly important in the early phases of defense against invading microbes because they are available within minutes to hours after the first contact with the pathogen. Moreover, the peptides exhibit a broad spectrum of activity that includes bacteria, fungi and certain enveloped viruses. Antimicrobial peptides, which numbered greater than 100 as recently as 1998, can be classified based on structural features (Hancock et al., 1995, Adv. Microb. Physiol. 372135-175; Boman 1995, Annu. Rev. Immunol. 13:61-92; and Lehrer and Ganz, 1996, Ann. Acad. Sci. 797:228-239).
However, many of these different structural classes of peptides share certain common properties. These include cationic charge, a broad spectrum of antimicrobial activity via selective discretion of target membranes, and encoding by genes which are expressed with tissue specificity.
One important element of the human innate immune defenses against microorganisms are small antimicrobial peptides known as defensins (Ganz and Lehrer, Curr Opin Immunol 10241-4, 1998). These small (30-50 aa) cationic peptides are found in a variety of mammalian myeloid and epithelial cells, and are bactericidal or bacteriostatic for a broad spectrum of microbes, including Mycobacterium tuberculosis (Ogata et al., Infect. Immun. 60:4720-4725, 1992; Miyakawa et al., Infect. Immun. 642926-932, 1996).
While defensins are found in rabbit (Patterson-Delafield et al., Infect Immun 312723-31, 1981) and bovine macrophages (Ryan et al., Infect. Immun. 662878-881, 1998), they are absent from human macrophages (present inventors' unpublished data). Although defensins have been proposed for use as therapeutics, chemical synthesis of these peptides is a challenge due to the complex pattern of disulfide bonds which stabilize the structure (Lauth et al., Insect Biochem Mol Biol 2821059-66, 1998), and recombinant methods do not produce sufficient yields (Harwig et al., Meth. in Enzymol. 236: 160-170, 1994; Valore and Ganz, Methods Mol Biol 782115-31, 1997).
Alternatively, using defensin proteins as antimicrobial agents was described using DNA to encode the defensins for intracellular expression in a murine macrophage cell line, which resulted in greater resistance to Histoplasma capsulatum (Couto et al., Injection & Immunity 62:2375-8, 1994). To date, however, there are very few reports of primary human macrophage transfection with DNA plasmids. Moreover, those which quantitate transfection efficiency report that only about 2% of the cells express the reporter gene (eGFP) (Simoes et al., J. Leulcoc Biol 651270-9, 1999; Van Tendeloo et al., Gene Ther 5:700-7, 1998; Weir and Meltzer, Cell Immunol 1482157-65, 1993).
On the other hand, the use of either bacteriostatic or bactericidal antibiotics is known to counteract bacterial infections to which humans are subjected, which may be different in nature. Although antibiotics are advantageous, they are not free from drawbacks because they generally involve side effects including imbalance of the bacterial flora of the skin, occurrence of allergic reactions, toxic effects in various districts of the organism, as well as intolerance in case of interaction with other substances, especially other drugs.
Furthermore, the overuse of antibiotics may result in the occurrence of an antibiotic resistance which can be acquired by the pathogenic agent, thus rendering ineffective the antibiotic therapy which nevertheless should be continued for a medium to long period to be successful.
In any case, the abuse of antibiotics may pose serious consequences to human organism which may even lead to death. To overcome the above drawbacks, alternative approaches to antibiotics have been investigated, particularly for the treatment of skin and mucous membranes suffering from diseases such as acne, which approaches showed the antimicrobial activity of a peptide.
Therefore, there is a need in the art for a feasible method of producing and using therapeutic antimicrobial compositions that do not naturally encounter the above problems.